CN104865348B - A kind of method of efficient detection nano-particle myocardial toxicity - Google Patents

A kind of method of efficient detection nano-particle myocardial toxicity Download PDF

Info

Publication number
CN104865348B
CN104865348B CN201510214824.3A CN201510214824A CN104865348B CN 104865348 B CN104865348 B CN 104865348B CN 201510214824 A CN201510214824 A CN 201510214824A CN 104865348 B CN104865348 B CN 104865348B
Authority
CN
China
Prior art keywords
nano
particle
gains
electrospinning
patterning
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201510214824.3A
Other languages
Chinese (zh)
Other versions
CN104865348A (en
Inventor
刘耀文
叶劲松
吴贺君
陈淑娟
何利
李美良
王淑瑶
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Yao Chen Industrial Development Co., Ltd.
Original Assignee
Sichuan Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sichuan Agricultural University filed Critical Sichuan Agricultural University
Priority to CN201510214824.3A priority Critical patent/CN104865348B/en
Publication of CN104865348A publication Critical patent/CN104865348A/en
Application granted granted Critical
Publication of CN104865348B publication Critical patent/CN104865348B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Medicinal Preparation (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A kind of method that the invention discloses efficient detection nano-particle myocardial toxicity, the method comprises the steps: 1) prepare patterned fibrous electrospinning reception plate, utilize optical etching technology and magnetically controlled DC sputtering technology, preparation has the reception plate of pattern metal silver layer;2) preparation patterning electrospinning fibre, utilizes step 1) gains to carry out electrostatic spinning;3) PDMS cavity is prepared;4) by step 2) and step 3) gains plasma treatment be attached;5) Primary mouse myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilize syringe pump that culture medium pumps into the cavity of step 4) gains, again nano-particle is pumped into, pump in nano-particle and in latter 20 days, record various myocardial toxicity index.The present invention can realize nano-particle myocardial toxicity efficient, high-precision detection, and Detection results is consistent with interior animal experiment;More quick relative to interior animal experiment.

Description

A kind of method of efficient detection nano-particle myocardial toxicity
Technical field
The invention belongs to nano-particle toxicity detection field, a kind of method being specifically related to efficient detection nano-particle myocardial toxicity.
Technical background
At biomedicine field, nano-particle is commonly used as the carrier of medicine conveying, when using nano-particle, often need to its toxicity be evaluated.
Heart is the vitals of human body, and can a kind of nano-particle carry out bio-medical, and cardiocellular toxicity is important investigation object by it.
In the prior art, the common method of the myocardial toxicity evaluating nano cell is carry out the zoopery of nano-particle toxicity.But carrying out zoopery, the required detection cycle is long, with often causing detection error due to the individual variation of laboratory animal.Prior art is used for substitute zooperal experiment in vitro, complex steps, it is easy to owing to operational error causes error, degree of accuracy is difficult to ensure that.
Summary of the invention
For the shortcoming of prior art, a kind of method that it is an object of the invention to provide efficient detection nano-particle myocardial toxicity, it is characterised in that described method comprises the steps:
1) prepare patterned fibrous electrospinning and receive plate: on insulating glass sheet, be first coated with positive-working photoresist, then cover lid layer photomask, utilize litho machine to perform etching;Again through deposition layer of metal silver on direct magnetic control technology sheet glass after etching, the shape of the argent deposited includes the one in square, rectangle, the length of side range for 20 μm-100 μm;Finally wash remaining positive-working photoresist;
2) preparation patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains to receive plate as electrospinning, prepared the patterning electrospinning fibre of square or rectangular composition by electrostatic spinning technique;The diameter controlling gained fiber is 100-300nm;
Described medical high polymer includes the one in polylactic acid, polycaprolactone, polyurethane, polyacrylonitrile, and described organic solvent includes at least one in acetone, dimethylformamide;
3) negative epoxy resin type nearultraviolet rays photoresist SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain width be 40-150 μm, height be that the SU-8 cuboid of 70-100 μm is as mould;Melted PDMS is placed on gained mould, after PDMS cools down, removes mould, obtain PDMS cavity;
4) by step 2) and step 3) gains at O2Or N2Atmosphere under, by Cement Composite Treated by Plasma 60 seconds so that step 2) and step 3) gains compact siro spinning technology;
5) by 5 × 106-1.0×107Individual mouse primary myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilizes syringe pump that culture medium pumps into the cavity of step 4) gains, and flow velocity is 0.2-0.3ml/h;Again nanoparticle suspension is pumped into after PBS, after nano-particle pumps into, record the change of the change of mouse primary myocardial cell jumping frequency rate, the enzyme work of superoxide dismutase, the activity of glutathion peroxidase, mda content, the damage of cell DNA, apoptosis rate, draw the toxicity of nano-particle;
Described nano-particle includes the one in nano ferriferrous oxide, nano titanium oxide, nano zine oxide, nano silicon, nano-Ag particles.
The present invention gropes to find by substantial amounts of experiment, it is the electrospinning fibre of 100-300nm when selecting diameter, and when the pattern of fibrous membrane is set to square or rectangular array, and arrange PDMS cavity be of a size of wide for 40-150 μm, high for 70-100 μm time, mouse primary myocardial cell can be made well to grow in cavity, and nano-particle is produced almost identical with zoopery toxic reaction.
The present invention can so that patterned fibrous and PDMS cavity and substrate of glass is seamless fits tightly, it is ensured that nano-particle completely with cells contacting, it is ensured that the degree of accuracy of detection.Simultaneously as patterned fibrous film is fitted with substrate and cavity closely so that the present invention can have good repetitive operation, saves the cost of detection.
It is essential that any predictable change made according to the present invention, as finely tuned pattern form and PDMS size, belong to protection scope of the present invention.
Special needs to be pointed out is, the present invention is applicable to the myocardial toxicity of detection nano-particle.General knowledge according to this area, it should be understood that the present invention detects suitable in the myocardial toxicity of any nano-particle.It should be understood that several nano-particle cited by the present invention are used only for proving that the present invention detects suitable in the myocardial toxicity of nano-particle, should not be construed as limitation of the invention.
In the present invention, the argent of step 1) deposition can be prepared as any type of array, and only needing the figure in array is square or rectangular of the present invention.
Preferably, in described step 1) deposition argent be shaped as square, the length of side range for 20 μm-100 μm.When being shaped as quadrate array of the argent deposited, primary cardiomyocytes has better growth conditions, is more beneficial for the detection of the myocardial toxicity of nano-particle.
Described step 2) in, when carrying out electrospinning, voltage is 15-25KV, and flow velocity is 0.5-1.0ml/h.
Preferably, described step 2) in, the diameter of fiber is 200nm.When the diameter of fiber used is 200nm, primary cardiomyocytes has better growth conditions, is more beneficial for the detection of the myocardial toxicity of nano-particle.
Described step 2) in medical high polymer be polylactic acid, described organic solvent is the mixed solution of acetone and dimethylformamide, and the volume ratio of acetone and dimethylformamide is 9:1.When fiber is acid fiber by polylactic, primary cardiomyocytes has better growth conditions, is more beneficial for the detection of the myocardial toxicity of nano-particle.
Preferably, the wide of the mould in described step 3) is 100 μm, and height is 80 μm.
It is furthermore preferred that the wide of mould in described step 3) is 50 μm, height is 70 μm.Detection tract is more little, more integrated, it is possible to achieve a detecting device has multiple tract, it is achieved the carrying out of more groups of parallel laboratory tests, more guarantees the degree of accuracy of detection.In the prior art, not yet it is found that prepare cavity so little, carry out myocardial toxicity detection based on electrospun fiber membrane.
In described step 5), the number of the primary cardiomyocytes for inoculating is 8 × 106Individual.
In described step 5), the flow velocity of culture medium is 0.25ml/h.
Beneficial effects of the present invention:
1, the present invention can realize nano-particle myocardial toxicity efficient, high-precision detection, and Detection results is consistent with interior animal experiment;More quick relative to interior animal experiment;
2, the present invention is raw materials used is easy to get, and cost is low, and technology maturation used is easily implemented, and has huge market application foreground.
Accompanying drawing explanation
Fig. 1 is gained patterned fibrous of the present invention.
Detailed description of the invention
The foregoing of the present invention is described in further detail by detailed description of the invention by the following examples, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below.
Embodiment 1
1) prepare patterned fibrous electrospinning and receive plate: on insulating glass sheet, be first coated with positive-working photoresist, then cover lid layer photomask, utilize litho machine to perform etching;Again through deposition layer of metal silver on direct magnetic control technology sheet glass after etching, the argent deposited be shaped as quadrate array, the length of side range for 20 μm;Finally wash remaining positive-working photoresist;
2) preparation patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains to receive plate as electrospinning, prepared the patterning electrospinning fibre of quadrate array by electrostatic spinning technique;The diameter controlling gained fiber is 100nm;
Described medical high polymer is polyacrylonitrile, and described organic solvent is acetone and dimethylformamide mixed solution (9:1v/v);
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 100 μm, height be that the SU-8 cuboid of 80 μm is as mould;Melted PDMS is placed on gained mould, after PDMS cools down, removes mould, obtain PDMS cavity;
4) passing through plasma treatment: open equipment vacuum room, loading good deposition prepared above has sheet glass and the PDMS cavity of patterning electrospinning fibre, opens RF driving source filament supply, opens O2:Gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly so that it is connect;
5) by 8 × 106Individual mouse primary myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilizes syringe pump that culture medium pumps into the cavity of step 4) gains, and flow velocity is 0.25ml/h;Again nanoparticle suspension is pumped into after PBS, after nano-particle pumps into, record the change of the cell jumping frequency rate change of mouse primary myocardial cell, the enzyme work of superoxide dismutase, the activity of glutathion peroxidase, mda content, the damage of cell DNA, apoptosis rate, draw the toxicity of nano-particle;
Described nano-particle includes nano-Ag particles.
Embodiment 2
1) prepare patterned fibrous electrospinning and receive plate: on insulating glass sheet, be first coated with positive-working photoresist, then cover lid layer photomask, utilize litho machine to perform etching;Again through deposition layer of metal silver on direct magnetic control technology sheet glass after etching, the argent deposited be shaped as rectangular array, the length of side is 20 μm and 70 μm, finally washes remaining positive-working photoresist;
2) preparation patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains to receive plate as electrospinning, prepared the patterning electrospinning fibre of rectangular array by electrostatic spinning technique;The diameter controlling gained fiber is 150nm;
Described medical high polymer includes polylactic acid, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 100 μm, height be that the SU-8 cuboid of 80 μm is as mould;Melted PDMS is placed on gained mould, after PDMS cools down, removes mould, obtain PDMS cavity;
4) passing through plasma treatment: open equipment vacuum room, loading good deposition prepared above has sheet glass and the PDMS cavity of patterning electrospinning fibre, opens RF driving source filament supply, opens N2Gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly so that it is connect;
5) by 8 × 106Individual mouse primary myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilizes syringe pump that culture medium pumps into the cavity of step 4) gains, and flow velocity is 0.25ml/h;Again nanoparticle suspension is pumped into after PBS, after nano-particle pumps into, record the change of the cell jumping frequency rate change of mouse primary myocardial cell, the enzyme work of superoxide dismutase, the activity of glutathion peroxidase, mda content, the damage of cell DNA, apoptosis rate, draw the toxicity of nano-particle;
Described nano-particle includes nano silicon.
Embodiment 3
1) prepare patterned fibrous electrospinning and receive plate: on insulating glass sheet, be first coated with positive-working photoresist, then cover lid layer photomask, utilize litho machine to perform etching;Again through deposition layer of metal silver on direct magnetic control technology sheet glass after etching, the argent deposited be shaped as quadrate array, the length of side is 50 μm;Finally wash remaining positive-working photoresist;
2) preparation patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains to receive plate as electrospinning, prepare square pattern electrospinning fibre by electrostatic spinning technique;The diameter controlling gained fiber is 200nm;
Described medical high polymer includes polyurethane, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 100 μm, height be that the SU-8 cuboid of 80 μm is as mould;Melted PDMS is placed on gained mould, after PDMS cools down, removes mould, obtain PDMS cavity;
4) passing through plasma treatment: open equipment vacuum room, loading good deposition prepared above has sheet glass and the PDMS cavity of patterning electrospinning fibre, opens RF driving source filament supply, opens N2Gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly so that it is connect;
5) by 8 × 106Individual mouse primary myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilizes syringe pump that culture medium pumps into the cavity of step 4) gains, and flow velocity is 0.2-0.3ml/h;Again nanoparticle suspension is pumped into after PBS, after nano-particle pumps into, record the change of the cell jumping frequency rate change of mouse primary myocardial cell, the enzyme work of superoxide dismutase, the activity of glutathion peroxidase, mda content, the damage of cell DNA, apoptosis rate, draw the toxicity of nano-particle;
Described nano-particle is nano zine oxide.
Embodiment 4
1) prepare patterned fibrous electrospinning and receive plate: on insulating glass sheet, be first coated with positive-working photoresist, then cover lid layer photomask, utilize litho machine to perform etching;Again through deposition layer of metal silver on direct magnetic control technology sheet glass after etching, the argent deposited be shaped as rectangular array, the length of side respectively 50 μm and 75 μm;Finally wash remaining positive-working photoresist;
2) preparation patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains to receive plate as electrospinning, by electrostatic spinning technique preparation patterning electrospinning fibre;The diameter controlling gained fiber is 250nm;
Described medical high polymer is polylactic acid, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 150 μm, height be that the SU-8 cuboid of 100 μm is as mould;Melted PDMS is placed on gained mould, after PDMS cools down, removes mould, obtain PDMS cavity;
4) passing through plasma treatment: open equipment vacuum room, loading good deposition prepared above has sheet glass and the PDMS cavity of patterning electrospinning fibre, opens RF driving source filament supply, opens O2Gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly so that it is connect;
5) by 1.0 × 107Individual mouse primary myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilizes syringe pump that culture medium pumps into the cavity of step 4) gains, and flow velocity is 0.3ml/h;Again nanoparticle suspension is pumped into after PBS, after nano-particle pumps into, record the change of the cell jumping frequency rate change of mouse primary myocardial cell, the enzyme work of superoxide dismutase, the activity of glutathion peroxidase, mda content, the damage of cell DNA, apoptosis rate, draw the toxicity of nano-particle;
Described nano-particle is nano ferriferrous oxide.
Embodiment 5
1) prepare patterned fibrous electrospinning and receive plate: on insulating glass sheet, be first coated with positive-working photoresist, then cover lid layer photomask, utilize litho machine to perform etching;Again through deposition layer of metal silver on direct magnetic control technology sheet glass after etching, the argent deposited be shaped as rectangular array, the length of side range for 20 μm and 100 μm;Finally wash remaining positive-working photoresist;
2) preparation patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains to receive plate as electrospinning, by electrostatic spinning technique preparation patterning electrospinning fibre;The diameter controlling gained fiber is 300nm;
Described medical high polymer is polycaprolactone, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 40 μm, height be that the SU-8 cuboid of 70 μm is as mould;Melted PDMS is placed on gained mould, after PDMS cools down, removes mould, obtain PDMS cavity;
4) passing through plasma treatment: open equipment vacuum room, loading good deposition prepared above has sheet glass and the PDMS cavity of patterning electrospinning fibre, opens RF driving source filament supply, opens O2Gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly so that it is connect;
5) by 5 × 106Individual mouse primary myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilizes syringe pump that culture medium pumps into the cavity of step 4) gains, and flow velocity is 0.2ml/h;Again nanoparticle suspension is pumped into after PBS, after nano-particle pumps into, record the change of the cell jumping frequency rate change of mouse primary myocardial cell, the enzyme work of superoxide dismutase, the activity of glutathion peroxidase, mda content, the damage of cell DNA, apoptosis rate, draw the toxicity of nano-particle;
Described nano-particle is nano titanium oxide.
Experimental example 1:
The method utilizing the embodiment of the present invention 1 carries out myocardial toxicity detection: when Primary mouse myocardial cell is in system of the present invention after In vitro culture 20 days, adding concentration is 80 μ g/ml, when particle diameter is about the nano-Ag particles of 80nm, after acting on 24 hours, the jumping frequency rate recording primary cardiomyocytes is 243 beats/min, the activity measuring its superoxide dismutase (SOD) is 10.44U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 47.14 μm of ol/gprot, the content of malonaldehyde (MDA) is 1.45nmol/mgprot, the damage ratio of DNA is 8.32%, apoptosis rate is 6.74%.
Experimental example 2:
The method utilizing the embodiment of the present invention 1 carries out myocardial toxicity detection: when Primary mouse myocardial cell is in system of the present invention after In vitro culture 15 days, adding concentration is 60 μ g/ml, when particle diameter is about the nano silicon of 70nm, after acting on 24 hours, the jumping frequency rate recording primary cardiomyocytes is 174 beats/min, the activity measuring its superoxide dismutase (SOD) is 35.56U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 54.03 μm of ol/gprot, the content of malonaldehyde (MDA) is 10.61nmol/mgprot, the damage ratio of DNA is 56.11%, apoptosis rate is 46.88%.
Experimental example 3:
The method utilizing the embodiment of the present invention 1 carries out myocardial toxicity detection: when Primary mouse myocardial cell is in system of the present invention after In vitro culture 10 days, adding concentration is 100 μ g/ml, when particle diameter is about the nano zine oxide of 50nm, after acting on 24 hours, the jumping frequency rate recording primary cardiomyocytes is 168 beats/min, the activity measuring its superoxide dismutase (SOD) is 54.32U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 67.35 μm of ol/gprot, the content of malonaldehyde (MDA) is 18.97nmol/mgprot, the damage ratio of DNA is 68.15%, apoptosis rate is 60.04%.
Experimental example 4:
The method utilizing the embodiment of the present invention 1 carries out myocardial toxicity detection: when Primary mouse myocardial cell is in system of the present invention after In vitro culture 10 days, adding concentration is 400 μ g/ml, when particle diameter is about the ferroso-ferric oxide of 30nm, after acting on 24 hours, the jumping frequency rate recording primary cardiomyocytes is 113 beats/min, the activity measuring its superoxide dismutase (SOD) is 87.46U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 79.57U/mgprot, the content of malonaldehyde (MDA) is 45.33nmol/mgprot, the damage ratio of DNA is 78.43%, apoptosis rate is 72.51%.
Experimental example 5:
The method utilizing the embodiment of the present invention 1 carries out myocardial toxicity detection: when Primary mouse myocardial cell is in system of the present invention after In vitro culture 10 days, adding concentration is 70 μ g/ml, when particle diameter is about the titanium dioxide of 10nm, after acting on 24 hours, the jumping frequency rate recording primary cardiomyocytes is 207 beats/min, the activity measuring its superoxide dismutase (SOD) is 43.15U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 13.28U/mgprot, the content of malonaldehyde (MDA) is 8.57nmol/mgprot, the damage ratio of DNA is 28.67%, apoptosis rate is 22.74%.
Contrast experiment's example 1:
Mice carried out tail vein injection experiment, implantation concentration is 80 μ g/ml, particle diameter is about the nanometer silver of 80nm and is about 5ml, effect body is after 24 hours, recording mouse heart jumping frequency rate is 266 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 15.65U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 49.58U/mgprot, the damage ratio that content is 2.37nmol/mgprot, DNA of malonaldehyde (MDA) is 3.58%, and apoptosis rate is 3.44%.
Contrast experiment's example 2:
Mice carried out tail vein injection experiment, implantation concentration is 60 μ g/ml, particle diameter is about the nano silicon of 70nm and is about 5ml, effect body is after 24 hours, recording mouse heart jumping frequency rate is 198 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 37.89U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 59.74U/mgprot, the damage ratio that content is 12.54nmol/mgprot, DNA of malonaldehyde (MDA) is 50.33%, and apoptosis rate is 40.24%.
Contrast experiment's example 3:
Mice carried out tail vein injection experiment, implantation concentration is 100 μ g/ml, particle diameter is about the nano zine oxide of 50nm and is about 5ml, effect body is after 24 hours, recording mouse heart jumping frequency rate is 185 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 57.67U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 70.25U/mgprot, the damage ratio that content is 21.07nmol/mgprot, DNA of malonaldehyde (MDA) is 59.18%, and apoptosis rate is 52.66%.
Contrast experiment's example 4:
Mice carried out tail vein injection experiment, implantation concentration is 400 μ g/ml, particle diameter is about the nano zine oxide of 30nm and is about 5ml, effect body is after 24 hours, recording mouse heart jumping frequency rate is 142 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 89.17U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 80.12U/mgprot, the damage ratio that content is 47.33nmol/mgprot, DNA of malonaldehyde (MDA) is 69.54%, and apoptosis rate is 65.37%.
Contrast experiment's example 5:
Mice carried out tail vein injection experiment, implantation concentration is 70 μ g/ml, particle diameter is about the nano titanium oxide of 10nm and is about 5ml, effect body is after 24 hours, recording mouse heart jumping frequency rate is 232 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 47.54U/mgprot, the activity of glutathion peroxidase (GSH-Px) is 16.35U/mgprot, the damage ratio that content is 9.67nmol/mgprot, DNA of malonaldehyde (MDA) is 21.93%, and apoptosis rate is 20.07%.
By above-mentioned experimental example 1-5 and contrast experiment example 1-5 it can be seen that in the scheming toxicity detection of nano-particle, the testing result of the present invention is similar to interior animal experiment result, can as the replacement scheme of interior animal experiment detection.It is essential that the present invention is more easy to control than experiment in vivo, and Financial cost and time cost lower, there is very good market application foreground.

Claims (9)

1. the method for an efficient detection nano-particle myocardial toxicity, it is characterised in that described method comprises the steps:
1) prepare patterned fibrous electrospinning and receive plate: on insulating glass sheet, be first coated with positive-working photoresist, then cover lid layer photomask, utilize litho machine to perform etching;Again through deposition layer of metal silver on direct magnetic control technology sheet glass after etching, the shape of the argent deposited includes the one in square, rectangle, the length of side range for 20 μm-100 μm;Finally wash remaining positive-working photoresist;
2) preparation patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains to receive plate as electrospinning, prepared the patterning electrospinning fibre of square or rectangular composition by electrostatic spinning technique;The diameter controlling gained fiber is 100-300nm;
Described medical high polymer includes the one in polylactic acid, polycaprolactone, polyurethane, polyacrylonitrile, and described organic solvent includes at least one in acetone, dimethylformamide;
3) negative epoxy resin type nearultraviolet rays photoresist SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain width be 40-150 μm, height be that the SU-8 cuboid of 70-100 μm is as mould;Melted PDMS is placed on gained mould, after PDMS cools down, removes mould, obtain PDMS cavity;
4) by step 2) and step 3) gains at O2Or N2Atmosphere under, by Cement Composite Treated by Plasma 60 seconds so that step 2) and step 3) gains compact siro spinning technology;
5) by 5 × 106-1.0×107Individual mouse primary myocardial cell is inoculated on the patterning electrospinning fibre in step 4) gains, utilizes syringe pump that culture medium pumps into the cavity of step 4) gains, and flow velocity is 0.2-0.3ml/h;Again nanoparticle suspension is pumped into after PBS, after nano-particle pumps into, record the change of the change of mouse primary myocardial cell jumping frequency rate, the enzyme work of superoxide dismutase, the activity of glutathion peroxidase, mda content, the damage of cell DNA, apoptosis rate, draw the toxicity of nano-particle;
Described nano-particle includes the one in nano ferriferrous oxide, nano titanium oxide, nano zine oxide, nano silicon, nano-Ag particles.
2. method according to claim 1, it is characterised in that in described step 1) deposition argent be shaped as square, the length of side range for 20 μm-100 μm.
3. method according to claim 1, it is characterised in that described step 2) in, when carrying out electrospinning, voltage is 15-25KV, and flow velocity is 0.5-1.0ml/h.
4. method according to claim 1, it is characterised in that described step 2) in, the diameter of fiber is 200nm.
5. method according to claim 1, it is characterised in that described step 2) in medical high polymer be polylactic acid, described organic solvent is the mixed solution of acetone and dimethylformamide, and the volume ratio of acetone and dimethylformamide is 9:1.
6. method according to claim 1, it is characterised in that the wide of the mould in described step 3) is 100 μm, and height is 80 μm.
7. method according to claim 1, it is characterised in that the wide of the mould in described step 3) is 50 μm, and height is 70 μm.
8. method according to claim 1, it is characterised in that in described step 5), the number of the Primary mouse myocardial cell for inoculating is 8 × 106Individual.
9. method according to claim 1, it is characterised in that in described step 5), the flow velocity of culture medium is 0.3ml/h.
CN201510214824.3A 2015-04-30 2015-04-30 A kind of method of efficient detection nano-particle myocardial toxicity Expired - Fee Related CN104865348B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510214824.3A CN104865348B (en) 2015-04-30 2015-04-30 A kind of method of efficient detection nano-particle myocardial toxicity

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510214824.3A CN104865348B (en) 2015-04-30 2015-04-30 A kind of method of efficient detection nano-particle myocardial toxicity

Publications (2)

Publication Number Publication Date
CN104865348A CN104865348A (en) 2015-08-26
CN104865348B true CN104865348B (en) 2016-07-06

Family

ID=53911326

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510214824.3A Expired - Fee Related CN104865348B (en) 2015-04-30 2015-04-30 A kind of method of efficient detection nano-particle myocardial toxicity

Country Status (1)

Country Link
CN (1) CN104865348B (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101413183A (en) * 2007-10-16 2009-04-22 国家纳米科学中心 Polymer electrostatic spinning film, preparation and use in biological detection
CN101481651A (en) * 2009-02-05 2009-07-15 上海纳米技术及应用国家工程研究中心有限公司 Metal nano material toxicity detection board and detection method thereof
CN102950036A (en) * 2012-11-01 2013-03-06 中国科学院大连化学物理研究所 Method for preparing microfluidic chip based on electrospinning template
CN102975318A (en) * 2012-11-06 2013-03-20 中国科学院大连化学物理研究所 Method for preparing PDMS chip including both square and arc-shaped channel
CN103173871A (en) * 2011-12-22 2013-06-26 中国科学院大连化学物理研究所 Method for producing nano electrospining with concentration gradient based on microfluidics technology
US20150060688A1 (en) * 2012-03-19 2015-03-05 Dalian University Of Technology Mems 2d air amplifier ion focusing device and manufacturing method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101413183A (en) * 2007-10-16 2009-04-22 国家纳米科学中心 Polymer electrostatic spinning film, preparation and use in biological detection
CN101481651A (en) * 2009-02-05 2009-07-15 上海纳米技术及应用国家工程研究中心有限公司 Metal nano material toxicity detection board and detection method thereof
CN103173871A (en) * 2011-12-22 2013-06-26 中国科学院大连化学物理研究所 Method for producing nano electrospining with concentration gradient based on microfluidics technology
US20150060688A1 (en) * 2012-03-19 2015-03-05 Dalian University Of Technology Mems 2d air amplifier ion focusing device and manufacturing method thereof
CN102950036A (en) * 2012-11-01 2013-03-06 中国科学院大连化学物理研究所 Method for preparing microfluidic chip based on electrospinning template
CN102975318A (en) * 2012-11-06 2013-03-20 中国科学院大连化学物理研究所 Method for preparing PDMS chip including both square and arc-shaped channel

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
Multiscale carbon structures fabricated by direct micropatterning of electrospun mats of US-8 photoresist nanofibers;Chandra S. Sharma等;《Langmuir》;20100113;第26卷(第4期);全文 *
Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments;Justin G. Teeguarden等;《Toxicological Sciences》;20061110;第95卷(第2期);全文 *
Preparation and characterization of novel bone scaffolds based on electrospun polycaprolactone fibers filled with nanoparticles;Patcharaporn Wutticharoenmongkol等;《Macromolecular Bioscience》;20060630(第6期);全文 *
基于玻璃基底微流控芯片的制备;程佳等;《生物医学工程研究》;20050630;第24卷(第3期);全文 *
微流控芯片上细胞的药物代谢及毒性研究;林金明;《中国毒理学会兽医毒理学与饲料毒理学学术讨论会暨兽医毒理专业委员会第4次全国代表大会》;20120901;摘要第1~11和26行 *
微流控芯片在细胞水平药物筛选中的研究进展;孙端平等;《中国新药杂志》;20130930;第22卷(第18期);全文 *
用于PDMS微芯片塑性成型的SU-8模具制备工艺的优化;陆振华等;《功能材料与器件学报》;20080630;第14卷(第3期);全文 *
聚乳酸-乙醇酸/纳米氧化锌复合电纺纤维装载亲疏水药物的控释及体外细胞毒性;曾莉等;《高等学校化学学报》;20140831;第35卷(第8期);全文 *
评价纳米颗粒细胞毒性的方法;李晓东,徐红;《中国实验诊断学》;20110131;第15卷(第1期);全文 *
银纳米颗粒对神经元的毒性研究;周国凤等;《北京生物医学工程》;20110630;第30卷(第3期);全文 *
静电纺丝法制备图案化微纳米纤维薄膜;龙云泽等;《青岛大学学报(自然科学版)》;20090930;第22卷(第3期);全文 *

Also Published As

Publication number Publication date
CN104865348A (en) 2015-08-26

Similar Documents

Publication Publication Date Title
Chen et al. Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis
Wang et al. Poking cells for efficient vector-free intracellular delivery
Krueger Beyond the shine: recent progress in applications of nanodiamond
Kinsella et al. DNA-templated magnetic nanowires with different compositions: fabrication and analysis
Scarpa et al. Biocompatibility studies of functionalized regioregular poly (3‐hexylthiophene) layers for sensing applications
Zhu et al. Recent advances in patterning natural polymers: from nanofabrication techniques to applications
JPH03502642A (en) Method and apparatus for detecting the effects of cellular agents on living cells
Tajiri et al. Regenerative potential of induced pluripotent stem cells derived from patients undergoing haemodialysis in kidney regeneration
CN109142712B (en) The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ
CN106633167B (en) It is a kind of for the imprinted material of specific recognition cell and its preparation and application
CN101196514B (en) Method for fixing cell in microchannel
CN104849439B (en) Efficient nanometer particle renal toxicity detection method
CN104164360B (en) Integrated microfluidic chip and for three-dimensional nodule location, build, recovery method
US20170239393A1 (en) Magnetic nanoparticle embedded nanofibrous membrane
Mohagheghian et al. Quantifying stiffness and forces of tumor colonies and embryos using a magnetic microrobot
CN109593719A (en) A method of ferric oxide nano particles are adsorbed on cell membrane
CN104865348B (en) A kind of method of efficient detection nano-particle myocardial toxicity
CN106932371B (en) Fluorescence imaging method of intracellular glutathione
CN110907416A (en) Circulating tumor cell detection device based on hollow nano needle tube electroporation system and detection method thereof
CN106963987A (en) A kind of conductive extracellular matrix laminated film obtained via cell sheets and preparation method thereof
Moghaddam et al. Fabrication of polyhydroxybutyrate (PHB)/γ-Fe2O3 nanocomposite film and its properties study
Ejidike et al. Role of Nanotechnology in Medicine: Opportunities and Challenges
Zhou et al. Actinomycin-X2-immobilized silk fibroin film with enhanced antimicrobial and wound healing activities
Wang et al. Graphene oxide-coated patterned silk fibroin films promote cell adhesion and induce cardiomyogenic differentiation of human mesenchymal stem cells
CN107496416B (en) EGCG aromatic medicine for treating Parkinson's disease and preparation method thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
EXSB Decision made by sipo to initiate substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CB03 Change of inventor or designer information
CB03 Change of inventor or designer information

Inventor after: Sun Dejie

Inventor before: Liu Yaowen

Inventor before: Ye Jinsong

Inventor before: Wu Hejun

Inventor before: Chen Shujuan

Inventor before: He Li

Inventor before: Li Meiliang

Inventor before: Wang Shuyao

TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20171225

Address after: 301700 Tianjin city Wuqing District Keihin Industrial Zone No. 7 Building 326 Keihin wisdom City

Patentee after: Tianjin Yao Chen Industrial Development Co., Ltd.

Address before: Ya'an City, Sichuan Province, 625014 new Kang Lu Yucheng District No. 46 (Sichuan Agricultural Uniersity)

Patentee before: Sichuan Agricultural University

CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20160706

Termination date: 20190430